Locations of disulfide bonds and free cysteines in the heavy and light chains of recombinant human factor VIII (antihemophilic factor A).

The locations of disulfide bonds and free cysteines in the heavy and light chains of recombinant human factor VIII were determined by sequence analysis of fragments produced by chemical and enzymatic digestions. The A1 and A2 domains of the heavy chain and the A3 domain of the light chain contain one free cysteine and two disulfide bonds, whereas the C1 and C2 domains of the light chain have one disulfide bond and no free cysteine. The positions of these disulfide bonds are conserved in factor V and ceruloplasmin except that the second disulfide bond in the A3 domain is missing in both factor V and ceruloplasmin. The positions of the three free cysteines of factor VIII are the same as three of the four cysteines present in ceruloplasmin. However, the positions of the free cysteines in factor VIII and ceruloplasmin are not conserved in factor V.     

The arrangement of intra- and intermolecular disulfide bonds in the carboxyterminal, non-collagenous aggregation and cross-linking domain of basement-membrane type IV collagen

The hexameric complex of globular domains of type IV collagen was isolated after collagenase digestion of human placenta and the different monomers and dimers present were chromatographically separated. The ratio of alpha 1(IV)NC1 to alpha 2(IV)NC1 was 2:1. About 50% of the NC1 domains were connected to dimers. Predominantly alpha 1-alpha 1 dimers were found. Only 12% were alpha 2-alpha 2 dimers and no alpha 1-alpha 2 dimers could be detected. The majority (88%) of the intermolecular bonds was found to be disulfide bridges. The remainder could not be cleaved by reduction. To elucidate the arrangement of the disulfide bonds, the unreduced alpha 1(IV)NC1 monomers were treated with cyanogen bromide, the disulfide-bridged peptides isolated and characterized by Edman degradation. Each of the two homologous subdomains within a monomer is stabilized by an identical set of three disulfide bonds. In subdomain I, cysteines at positions 20 and 53 are connected with the C-terminal cysteine pair 108 and 111. Thus formed, the disulfide knot stabilizes two interconnected loops of 32 and 54 residues, respectively. A smaller loop of five residues occurs due to a disulfide bond between the cysteines 65 and 71. A similar disulfide arrangement is indicated for subdomain II which is separated from subdomain I by a segment of 20 amino acid residues. The same arrangement of disulfide bonds has been strongly suggested for the alpha 2(IV)NC1 monomer by the isolation and characterization of its disulfide-bridged tryptic fragments. Similar investigations on the dimeric alpha 1(IV)NC1 domain established the arrangement of the intermolecular disulfide bonds. They are formed by a complete disulfide exchange between corresponding disulfide knots of two monomeric NC1 domains.     

Nonnative interactions between cysteines direct productive assembly of P22 tailspike protein

Nonnative disulfide bond formation can play a critical role in the assembly of disulfide bonded proteins. During the folding and assembly of the P22 tailspike protein, nonnative disulfide bonds form both in vivo and in vitro. However, the mechanism and identity of cysteine disulfide pairs remains elusive, particularly for P22 tailspike, which contains no disulfide bonds in its native, functional form. Understanding the interactions between cysteine residues is important for developing a mechanistic model for the role of nonnative cysteines in P22 tailspike assembly. Prior in vivo studies have suggested that cysteines 496, 613, and 635 are the most likely site for sulfhydryl reactivity. Here we demonstrate that these three cysteines are critical for efficient assembly of tailspike trimers, and that interactions between cysteine pairs lead to productive assembly of native tailspike.     

Identification and function of disulfide bridges in the extracellular domains of the angiotensin II type 2 receptor.

The angiotensin II (AngII) receptor family is comprised of two subtypes, type 1 (AT(1)) and type 2 (AT(2)). Although sharing low homology (only 34%), mutagenesis has identified some key residues that are conserved between both subtypes, including four extracellular cysteines. Previous AT(1) mutagenesis demonstrated that the cysteines form two disulfide bonds, one linking the first and second extracellular loops and another connecting the amino terminus to the third extracellular loop. The importance of these AT(1) disulfides in ligand binding is supported by the effect of dithiothreitol (DTT). DTT breaks disulfide bonds, thereby strongly inhibiting ligand binding in AT(1) receptors. Despite retaining the same cysteines, AT(2) receptor ligand binding is paradoxically enhanced by DTT. Thus, we constructed a series of AT(2) cysteine mutations, either individually or paired, to establish the role of the cysteines and the source of DTT's effects. The AT(2) cysteine mutants surprisingly confirmed that the cysteines form disulfide bonds in the same manner as in the AT(1) subtype. However, breaking the AT(2) disulfide bridges yielded two responses. As in AT(1) receptors, mutations disrupting the disulfide bond between the first and second extracellular loops reduced AT(2) binding by 4-fold. In contrast, mutations breaking the disulfide bridge between the amino terminus and the third extracellular loop increased AT(2) binding, mimicking DTT's effect on this subtype. Further analysis of AT(1)/AT(2) chimeric exchange mutants of these domains suggested that the AT(2) amino terminus and third extracellular loop may possess latent binding epitopes that are only uncovered after DTT exposure.     

Formation of Two Intramolecular Disulfide Bonds Is Necessary for ApoA-I-dependent Cholesterol Efflux Mediated by ABCA1

ABCA1 plays a major role in cholesterol homeostasis and high density lipoprotein (HDL) metabolism. ABCA1 contains disulfide bond(s) between its N- and C-terminal halves, but it remains unclear whether disulfide bond formation is important for the functions of ABCA1 and which cysteines are involved in disulfide bond formation. To answer these questions, we constructed >30 ABCA1 mutants in which 16 extracellular domain (ECD) cysteines were replaced with serines and examined disulfide bond formation, apoA-I binding, and HDL formation in these mutants. From the single cysteine replacements, two cysteines (Cys⁷⁵ and Cys³⁰⁹) in ECD1 were found to be essential for apoA-I binding. In contrast, in ECD2, only Cys¹⁴⁷⁷ was found to be essential for HDL formation, and no single cysteine replacement impaired apoA-I binding. The concurrent replacement of two cysteines, Cys¹⁴⁶³ and Cys¹⁴⁶⁵, impaired apoA-I binding and HDL formation, suggesting that four of five extracellular cysteines (Cys⁷⁵, Cys³⁰⁹, Cys¹⁴⁶³, Cys¹⁴⁶⁵, and Cys¹⁴⁷⁷) are involved in these functions of ABCA1. Trypsin digestion experiments suggested that one disulfide bond is not sufficient and that two intramolecular disulfide bonds (between Cys⁷⁵ and Cys³⁰⁹ in ECD1 and either Cys¹⁴⁶³ or Cys¹⁴⁶⁵ and Cys¹⁴⁷⁷ in ECD2) are required for ABCA1 to be fully functional.Maintenance of cellular cholesterol homeostasis is important for normal human physiology; its disruption can lead to a variety of pathological conditions, including cardiovascular disease (1). ABCA1 (ATP-binding cassette protein A1), a key factor in cholesterol homeostasis, mediates the secretion of cellular free cholesterol and phospholipids to an extracellular acceptor, apoA-I, to form high density lipoprotein (HDL)² (2, 3). HDL formation is the only known pathway for the elimination of excess cholesterol from peripheral cells. Defects in ABCA1 cause Tangier disease (4–6), in which patients have a near absence of circulating HDL, prominent cholesterol ester accumulation in tissue macrophages, and premature atherosclerotic vascular disease (1, 7).ABCA1 is a member of the ABCA subclass of ABC transporters, which contain the basic architecture of the “full-length” ABC transporters organized into two tandemly arranged halves. Each half contains several transmembrane α-helices (TMs), which provide a translocation pathway, followed by a cytoplasmic nucleotide-binding domain, which hydrolyze ATP. In the case of “half-size” ABC transporters, such as ABCG1, ABCD1, TAP1/TAP2 (transporter associated with antigen processing), and the bacterial homolog Sav1866, they dimerize to form the full transporter. Crystallographic analysis of the bacterial homolog Sav1866 revealed that the TMs of one subunit are closely related to the TMs of the other subunit, forming two “wings” in an outward-facing conformation (8).When ABCA1 is partially digested by trypsin, ABCA1 is cleaved at site A, just C-terminal to TM6, and at site B, just N-terminal to TM7, to produce four fragments of 170 and 150 kDa and subsequently of 125 and 110 kDa (Fig. 1A) (9). When these fragments are analyzed by SDS-PAGE under nonreducing conditions, they co-migrate with undigested ABCA1. These results suggest that the N- and C-terminal halves of ABCA1 are connected by disulfide bond(s), as reported for ABCA4 (ABCR) (10). The ABCA subclass is distinguished from other ABC transporter subclasses by the presence of large extracellular domains (ECDs) (Fig. 1A) (11, 12). ECD1 and ECD2 of ABCA1 contain nine and five cysteine residues, respectively, and each connecting loop between TM5 and TM6 and between TM11 and TM12 contains a cysteine residue. These cysteine residues were assigned numbers, C1 to C16, based on their distance from the N terminus (Fig. 1A). These cysteine residues are well conserved among ABCA1, ABCA4, and ABCA7 (Fig. 1B). All of the cysteine residues in ECD1 are conserved between ABCA1 and ABCA4, and seven cysteine residues (except C4 and C5) are conserved in ABCA7. All five of the cysteine residues in ECD2 are conserved between ABCA1 and ABCA4, and three cysteine residues (except C11 and C12) are conserved in ABCA7. Because ABCA7, like ABCA1, mediates apoA-I-dependent lipid efflux (13, 14), conserved cysteine residues might be important for its function. Indeed, the Tangier disease mutation C1477R has been reported to abolish apoA-I binding and HDL formation (15–17), and several missense mutations in cysteine residues within ECD1 (C54Y, C75G) and ECD2 (C1488R, C1490Y) of ABCA4 have been linked to Stargardt disease (18–21). It remains unclear, however, whether disulfide bond formation is important for the proper folding and/or the functions of ABCA subclass proteins.Open in a separate windowFIGURE 1.Structural features of ABCA1. A, topological model for human ABCA1. ABCA1 consists of 12 transmembrane α-helices (TM1–TM12) and two large ECDs. ECD1 and ECD2 contain nine and five cysteine residues, respectively, and each connecting loop between TM5 and TM6 and between TM11 and TM12 contains a cysteine residue. These cysteine residues were assigned numbers, from C1 to C16, based on their distance from the N terminus. ABCA1 is cleaved at sites A and B by limited trypsin digestion. B, amino acid sequence alignment of ECDs of human ABCA1, ABCA4, and ABCA7. Conserved cysteine residues are indicated in black boxes.In this study, we analyzed which cysteine residues are involved in disulfide bond formation and examined whether disulfide bond formation is necessary for the functions of ABCA1. Cysteine substitution experiments suggested that two disulfide bonds are formed between C2 and C6 in ECD1 and between either C13 or C14 and C15 in ECD2 and that this two-disulfide bond formation is necessary for apoA-I-dependent cholesterol efflux by ABCA1.     

The α-Proteobacteria Wolbachia pipientis Protein Disulfide Machinery Has a Regulatory Mechanism Absent in γ-Proteobacteria

The α-proteobacterium Wolbachia pipientis infects more than 65% of insect species worldwide and manipulates the host reproductive machinery to enable its own survival. It can live in mutualistic relationships with hosts that cause human disease, including mosquitoes that carry the Dengue virus. Like many other bacteria, Wolbachia contains disulfide bond forming (Dsb) proteins that introduce disulfide bonds into secreted effector proteins. The genome of the Wolbachia strain wMel encodes two DsbA-like proteins sharing just 21% sequence identity to each other, α-DsbA1 and α-DsbA2, and an integral membrane protein, α-DsbB. α-DsbA1 and α-DsbA2 both have a Cys-X-X-Cys active site that, by analogy with Escherichia coli DsbA, would need to be oxidized to the disulfide form to serve as a disulfide bond donor toward substrate proteins. Here we show that the integral membrane protein α-DsbB oxidizes α-DsbA1, but not α-DsbA2. The interaction between α-DsbA1 and α-DsbB is very specific, involving four essential cysteines located in the two periplasmic loops of α-DsbB. In the electron flow cascade, oxidation of α-DsbA1 by α-DsbB is initiated by an oxidizing quinone cofactor that interacts with the cysteine pair in the first periplasmic loop. Oxidizing power is transferred to the second cysteine pair, which directly interacts with α-DsbA1. This reaction is inhibited by a non-catalytic disulfide present in α-DsbA1, conserved in other α-proteobacterial DsbAs but not in γ-proteobacterial DsbAs. This is the first characterization of the integral membrane protein α-DsbB from Wolbachia and reveals that the non-catalytic cysteines of α-DsbA1 regulate the redox relay system in cooperation with α-DsbB.     

Role of individual cysteine residues and disulfide bonds in the structure and function of Aspergillus ribonucleolytic toxin restrictocin.

Restrictocin, produced by the fungus Aspergillus restrictus, belongs to the group of ribonucleolytic toxins called ribotoxins. It specifically cleaves a single phosphodiester bond in a conserved stem and loop structure in the 28S rRNA of large ribosomal subunit and potently inhibits eukaryotic protein synthesis. Restrictocin contains 149 amino acid residues and includes four cysteines at positions 5, 75, 131, and 147. These cysteine residues are involved in the formation of two disulfide bonds, one between Cys 5 and Cys 147 and another between Cys 75 and Cys 131. In the current study, all four cysteine residues were changed to alanine individually and in different combinations by site-directed mutagenesis so as to remove one or both the disulfides. The mutants were expressed and purified from Escherichia coli. Removal of any cysteine or any one of the disulfide bonds individually did not affect the ability of the toxin to specifically cleave the 28S rRNA or to inhibit protein synthesis in vitro. However, the toxin without both disulfide bonds completely lost both ribonucleolytic and protein synthesis inhibition activities. The active mutants, containing only one disulfide bond, exhibited relatively high susceptibility to trypsin digestion. Thus, none of the four cysteine residues is directly involved in restrictocin catalysis; however, the presence of any one of the two disulfide bonds is absolutely essential and sufficient to maintain the enzymatically active conformation of restrictocin. For maintenance of the unique stability displayed by the native toxin, both disulfide bonds are required.     

Structure-Function Analysis of CCL28 in the Development of Post-viral Asthma

CCL28 is a human chemokine constitutively expressed by epithelial cells in diverse mucosal tissues and is known to attract a variety of immune cell types including T-cell subsets and eosinophils. Elevated levels of CCL28 have been found in the airways of individuals with asthma, and previous studies have indicated that CCL28 plays a vital role in the acute development of post-viral asthma. Our study builds on this, demonstrating that CCL28 is also important in the chronic post-viral asthma phenotype. In the absence of a viral infection, we also demonstrate that CCL28 is both necessary and sufficient for induction of asthma pathology. Additionally, we present the first effort aimed at elucidating the structural features of CCL28. Chemokines are defined by a conserved tertiary structure composed of a three-stranded β-sheet and a C-terminal α-helix constrained by two disulfide bonds. In addition to the four disulfide bond-forming cysteine residues that define the traditional chemokine fold, CCL28 possesses two additional cysteine residues that form a third disulfide bond. If all disulfide bonds are disrupted, recombinant human CCL28 is no longer able to drive mouse CD4+ T-cell chemotaxis or in vivo airway hyper-reactivity, indicating that the conserved chemokine fold is necessary for its biologic activity. Due to the intimate relationship between CCL28 and asthma pathology, it is clear that CCL28 presents a novel target for the development of alternative asthma therapeutics.     

Disulfide bond formation during the folding of influenza virus hemagglutinin

To study the importance of individual sulfhydryl residues during the folding and assembly in vivo of influenza virus hemagglutinin (HA), we have constructed and expressed a series of mutant HA proteins in which cysteines involved in three disulfide bonds have been substituted by serine residues. Investigations of the structure and intracellular transport of the mutant proteins indicate that (a) cysteine residues in the ectodomain are essential both for efficient folding of HA and for stabilization of the folded molecule; (b) cysteine residues in the globular portion of the ectodomain are likely to form native disulfide bonds rapidly and directly, without involvement of intermediate, nonnative linkages; and (c) cysteine residues in the stalk portion of the ectodomain also appear not to form intermediate disulfide bonds, even though they have the opportunity to do so, being separated from their correct partners by hundreds of amino acids including two or more other sulfhydryl residues. We propose a role for the cellular protein BiP in shielding the cysteine residues of the stalk domain during the folding process, thus preventing them from forming intermediate, nonnative disulfide bonds.     

Paired cysteine mutagenesis to establish the pattern of disulfide bonds in the functional intact secretin receptor

The amino-terminal domain of class B G protein-coupled receptors contains six conserved cysteine residues involved in structurally and functionally critical disulfide bonds. The mapping of these bonds has been unclear, with one pattern based on biochemical and NMR structural characterizations of refolded, nonglycosylated amino-terminal fragments, and another pattern derived from functional characterizations of intact receptors having paired cysteine mutations. In the present study, we determined the disulfide bonding pattern of the prototypic class B secretin receptor by applying the same paired cysteine mutagenesis approach and confirming the predicted bonding pattern with proteolytic cleavage of intact functional receptor. As expected, systematic mutation to serine of the six conserved cysteine residues within this region of the secretin receptor singly and in pairs resulted in loss of function of most constructs. Notable exceptions were single mutations of the 4th and 6th cysteine residues and paired mutations involving the 1st and 3rd, 2nd and 5th, and 4th and 6th conserved cysteines, with secretin eliciting statistically significant cAMP responses above basal levels of activation for each of these constructs. Immunofluorescence microscopy confirmed similar levels of plasma membrane expression for each of the mutated receptors. Furthermore, cyanogen bromide cleaved a series of wild type and mutant secretin receptors, yielding patterns that agreed with our paired cysteine mutagenesis results. In conclusion, these data suggest the same pattern of disulfide bonding as that predicted previously by NMR and thus support a consistent pattern of amino-terminal disulfide bonds in class B G protein-coupled receptors.     

Functional rescue of the nephrogenic diabetes insipidus-causing vasopressin V2 receptor mutants G185C and R202C by a second site suppressor mutation

Mutations in the gene of the G protein-coupled vasopressin V2 receptor (V2 receptor) cause X-linked nephrogenic diabetes insipidus (NDI). Most of the missense mutations on the extracellular face of the receptor introduce additional cysteine residues. Several groups have proposed that these residues might disrupt the conserved disulfide bond of the V2 receptor. To test this hypothesis, we first calculated a structure model of the extracellular receptor domains. The model suggests that the additional cysteine residues may form a second disulfide bond with the free, nonconserved extracellular cysteine residue Cys-195 rather than impairing the conserved bond. To address this question experimentally, we used the NDI-causing mutant receptors G185C and R202C. Their Cys-195 residues were replaced by alanine to eliminate the hypothetical second disulfide bonds. This second site mutation led to functional rescue of both NDI-causing mutant receptors, strongly suggesting that the second disulfide bonds are indeed formed. Furthermore we show that residue Cys-195, which is sensitive to "additional cysteine" mutations, is not conserved among the V2 receptors of other species and that the presence of an uneven number of extracellular cysteine residues, as in the human V2 receptor, is rare among class I G protein-coupled receptors.     

Disulfide bond structure determination and biochemical analysis of glycoprotein C from herpes simplex virus.

A biochemical analysis of glycoprotein C (gC of herpes simplex virus was undertaken to further characterize the structure of the glycoprotein and to determine its disulfide bond arrangement. We used three recombinant forms of gC, gC1(457t), gC1(delta33-123t), and gC2(426t), each truncated prior to the transmembrane region. The proteins were expressed and secreted by using a baculovirus expression system and have been shown to bind to monoclonal antibodies which recognize discontinuous epitopes and to complement component C3b in a dose-dependent manner. We confirmed the N-terminal residues of each mature protein by Edman degradation and confirmed the internal deletion in gC1(delta33-123t). The molecular weight and extent of glycosylation of gC1 (457t), gC1(delta33-123t), and gC2(426t) were determined by treating each protein with endoglycosidases and then subjecting it to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric analysis. The data indicate that eight to nine of the predicted N-linked oligosaccharide sites on gC1(457t) are occupied by glycans of approximately 1,000 Da. In addition, O-linked oligosaccharides are present on gC1(457t), primarily localized to the N-terminal region (amino acids [aa] 33 to 123) of the protein. gC2(426t) contains N-linked oligosaccharides, but no O-linked oligosaccharides were detected. To determine the disulfide bond arrangement of the eight cysteines of gC1(457t),the protein was cleaved with cyanogen bromide. SDS-PAGE analysis followed by Edman degradation identified three cysteine-containing fragments which are not connected by disulfide linkages. Chemical modification of cysteines combined with matrix-assisted laser desorption ionization mass spectrometry identified disulfide bonds between cysteine 1 (aa 127) and cysteine 2 (aa 144) and between cysteine 3 (aa 286) and cysteine 4 (aa 347). Further proteolysis of the cyanogen bromide-generated fragment containing cysteine 5 through cysteine 8, combined with mass spectrometry and Edman degradation, showed that disulfide bonds link cysteine 5 (aa 386) to cysteine 8 (aa 442) and cysteine 6 (aa 390) to cysteine 7 (aa 419). A similar disulfide bond arrangement is postulated to exist in gC homologs from other herpesviruses.     

Characterization and analysis of scFv-IgG bispecific antibody size variants

ABSTRACT

Bispecific antibodies are an emergent class of biologics that is of increasing interest for therapeutic applications. In one bispecific antibody format, single-chain variable fragments (scFv) are linked to or inserted in different locations of an intact immunoglobulin G (IgG) molecule to confer dual epitope binding. To improve biochemical stability, cysteine residues are often engineered on the heavy- and light-chain regions of the scFv to form an intrachain disulfide bond. Although this disulfide bond often improves stability, it can also introduce unexpected challenges to manufacturing or development. We report size variants that were observed for an appended scFv-IgG bispecific antibody. Structural characterization studies showed that the size variants resulted from the engineered disulfide bond on the scFv, whereby the engineered disulfide was found to be either open or unable to form an intrachain disulfide bond due to cysteinylation or glutathionylation of the cysteines. Furthermore, the scFv engineered cysteines also formed intermolecular disulfide bonds, leading to the formation of highly stable dimers and aggregates. Because both the monomer variants and dimers showed lower bioactivity, they were considered to be product-related impurities that must be monitored and controlled. To this end, we developed and optimized a robust, precise, and accurate high-resolution size-exclusion chromatographic method, using a statistical design-of-experiments methodology.     

Four cysteines of the membrane protein DsbB act in concert to oxidize its substrate DsbA

Protein disulfide bond formation in Escherichia coli is catalyzed by the periplasmic protein DsbA. A cytoplasmic membrane protein DsbB maintains DsbA in the oxidized state by transferring electrons from DsbA to quinones in the respiratory chain. Here we show that DsbB activity can be reconstituted by co-expression of N- and C-terminal fragments of the protein, each containing one of its redox-active disulfide bonds. This system has allowed us (i) to demonstrate that the two DsbB redox centers interact directly through a disulfide bond formed between the two DsbB domains and (ii) to identify the specific cysteine residues involved in this covalent interaction. Moreover, we are able to capture an intermediate in the process of electron transfer from one redox center to the other. These results lead us to propose a model that describes how the cysteines cooperate in the early stages of oxidation of DsbA. DsbB appears to adopt a novel mechanism to oxidize DsbA, using its two pairs of cysteines in a coordinated reaction to accept electrons from the active cysteines in DsbA.     

Human RNase H1 activity is regulated by a unique redox switch formed between adjacent cysteines

Human RNase H1 is active only under reduced conditions. Oxidation as well as N-ethylmaleimide (NEM) treatment of human RNase H1 ablates the cleavage activity. The oxidized and NEM alkylated forms of human RNase H1 exhibited binding affinities for the heteroduplex substrate comparable with the reduced form of the enzyme. Mutants of human RNase H1 in which the cysteines were either deleted or substituted with alanine exhibited cleavage rates comparable with the reduced form of the enzyme, suggesting that the cysteine residues were not required for catalysis. The cysteine residues responsible for the observed redox-dependent activity of human RNase H1 were determined by site-directed mutagenesis to involve Cys(147) and Cys(148). The redox states of the Cys(147) and Cys(148) residues were determined by digesting the reduced, oxidized, and NEM-treated forms of human RNase H1 with trypsin and analyzing the cysteine containing tryptic fragments by micro high performance liquid chromatography-electrospray ionization-Fourier transform ion cyclotron mass spectrometry. The tryptic fragment Asp(131)-Arg(153) containing Cys(147) and Cys(148) was identified. The mass spectra for the Asp(131)-Arg(153) peptides from the oxidized and reduced forms of human RNase H1 in the presence and absence of NEM showed peptide masses consistent with the formation of a disulfide bond between Cys(147) and Cys(148). These data show that the formation of a disulfide bond between adjacent Cys(147) and Cys(148) residues results in an inactive enzyme conformation and provides further insights into the interaction between human RNase H1 and the heteroduplex substrate.     

The redox state of the cell regulates the ligand binding affinity of human neuroglobin and cytoglobin

Neuroglobin and cytoglobin reversibly bind oxygen in competition with the distal histidine, and the observed oxygen affinity therefore depends on the properties of both ligands. In the absence of an external ligand, the iron atom of these globins is hexacoordinated. There are three cysteine residues in human neuroglobin; those at positions CD7 and D5 are sufficiently close to form an internal disulfide bond. Both cysteine residues in cytoglobin, although localized in other positions than in human neuroglobin, may form a disulfide bond as well. The existence and position of these disulfide bonds was demonstrated by mass spectrometry and thiol accessibility studies. Mutation of the cysteines involved, or the use of reducing agents to break the S-S bond, led to a decrease in the observed oxygen affinity of human neuroglobin by an order of magnitude. The critical parameter is the histidine dissociation rate, which changes by about a factor of 10. The same effect is observed with human cytoglobin, although to a much lesser extent (less than a factor of 2). These results suggest a novel mechanism for the regulation of oxygen binding; contact with an appropriate electron donor would provoke the release of oxygen. Hence the oxygen affinity would be directly linked to the redox state of the cell.     

Predicting disulfide bond connectivity in proteins by correlated mutations analysis

MOTIVATION: Prediction of disulfide bond connectivity facilitates structural and functional annotation of proteins. Previous studies suggest that cysteines of a disulfide bond mutate in a correlated manner. RESULTS: We developed a method that analyzes correlated mutation patterns in multiple sequence alignments in order to predict disulfide bond connectivity. Proteins with known experimental structures and varying numbers of disulfide bonds, and that spanned various evolutionary distances, were aligned. We observed frequent variation of disulfide bond connectivity within members of the same protein families, and it was also observed that in 99% of the cases, cysteine pairs forming non-conserved disulfide bonds mutated in concert. Our data support the notion that substitution of a cysteine in a disulfide bond prompts the substitution of its cysteine partner and that oxidized cysteines appear in pairs. The method we developed predicts disulfide bond connectivity patterns with accuracies of 73, 69 and 61% for proteins with two, three and four disulfide bonds, respectively.     

Disulfide bond in Pseudomonas aeruginosa lipase stabilizes the structure but is not required for interaction with its foldase

Pseudomonas aeruginosa secretes a 29-kDa lipase which is dependent for folding on the presence of the lipase-specific foldase Lif. The lipase contains two cysteine residues which form an intramolecular disulfide bond. Variant lipases with either one or both cysteines replaced by serines showed severely reduced levels of extracellular lipase activity, indicating the importance of the disulfide bond for secretion of lipase through the outer membrane. Wild-type and variant lipase genes fused to the signal sequence of pectate lyase from Erwinia carotovora were expressed in Escherichia coli, denatured by treatment with urea, and subsequently refolded in vitro. Enzymatically active lipase was obtained irrespective of the presence or absence of the disulfide bond, suggesting that the disulfide bond is required neither for correct folding nor for the interaction with the lipase-specific foldase. However, cysteine-to-serine variants were more readily denatured by treatment at elevated temperatures and more susceptible to proteolytic degradation by cell lysates of P. aeruginosa. These results indicate a stabilizing function of the disulfide bond for the active conformation of lipase. This conclusion was supported by the finding that the disulfide bond function could partly be substituted by a salt bridge constructed by changing the two cysteine residues to arginine and aspartate, respectively.     

Specific cysteines in beta3 are involved in disulfide bond exchange-dependent and -independent activation of alphaIIbbeta3

Disulfide bond exchange among cysteine residues in epidermal growth factor (EGF)-like domains of beta3 was suggested to be involved in activation of alphaIIbbeta3. To investigate the role of specific beta3 cysteines in alphaIIbbeta3 expression and activation, we expressed in baby hamster kidney cells normal alphaIIb with normal beta3 or beta3 with single or double cysteine substitutions of nine disulfide bonds in EGF-3, EGF-4, and beta-tail domains and assessed alphaIIbbeta3 surface expression and activation state by flow cytometry using P2 or PAC-1 antibodies, respectively. Most mutants displayed reduced surface expression of alphaIIbbeta3. Disruptions of disulfide bonds in EGF-3 yielded constitutively active alphaIIbbeta3, implying that these bonds stabilize the inactive alphaIIbbeta3 conformer. Mutants of the Cys-567-Cys-581 bond in EGF-4 were inactive even after exposure to alphaIIbbeta3-activating antibodies, indicating that this bond is necessary for activating alphaIIbbeta3. Disrupting Cys-560-Cys-583 in the EGF-3/EGF-4 or Cys-608-Cys-655 in beta-tail domain resulted in alphaIIbbeta3 activation only when Cys-560 or Cys-655 of each pair was mutated but not when their partners (Cys-583, Cys-608) or both cysteines were mutated, suggesting that free sulfhydryls of Cys-583 and Cys-608 participate in alphaIIbbeta3 activation by a disulfide bond exchange-dependent mechanism. The free sulfhydryl blocker dithiobisnitrobenzoic acid inhibited 70% of anti-LIBS6 antibody-induced activation of wild-type alphaIIbbeta3 and had a smaller effect on mutants, implicating disulfide bond exchange-dependent and -independent mechanisms in alphaIIbbeta3 activation. These data suggest that different disulfide bonds in beta3 EGF and beta-tail domains play variable structural and regulatory roles in alphaIIbbeta3.